Quantifying aluminum toxicity effects on corn phenotype using advanced imaging technologies

Abstract Soil acidity (pH <5.5) limits agricultural production due to aluminum (Al) toxicity. The primary target of Al toxicity is the plant root. However, symptoms can be observed on the shoots. This study aims to determine the potential use of chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning technology to quantify the effects of Al toxicity on corn. Corn seedlings were grown for 13 days in nutrient solutions (pH 4.0) with four Al treatments: 50, 100, 200, and 400 μM and a control (0 μM AlCl3 L−1). During the experiment, four measurements were performed: four (MT1), six (MT2), 11 (MT3), and 13 (MT4) days after the application of Al treatments. The most sensitive traits affected by Al toxicity were the reduction of plant growth and increased reflectance in the visible wavelength (affected at MT1). The reflectance of red wavelengths increased more significantly compared to near‐infrared and green wavelengths, leading to a decrease in the normalized difference vegetation index and the Green Leaf Index. The most sensitive chlorophyll fluorescence traits, effective quantum yield of PSII, and photochemical quenching coefficient were affected after prolonged Al exposure (MT3). This study demonstrates the usability of selected phenotypic traits in remote sensing studies to map Al‐toxic soils and in high‐throughput phenotyping studies to screen Al‐tolerant genotypes.

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These points include: 1. Provide appropriate details in the methods and figure legends, as appropriate, regarding both biological and technical replication (See Reviewer 1 comments).2. Expand the Discussion to cover the potentially unexpected observation that NPQ decreases with increasing AI concentration at the first two time points (MT1 and MT2), but these parameters both decrease at increasing Al concentration at MT3 and MT4.rETR shows a similar change in Al concentration response -in this case, increasing at MT1 and MT2, and decreasing with increasing Al concentration in MT3 and MT4.Although these opposite trends for both traits were reported in the Results they are not discussed or put in context of what is reported in the literature.See Reviewer 1 comments.3. The authors conclude that these above-ground Al-induced phenotypes identified by highthroughput imaging phenotyping could be used "in remote sensing studies to map Al-toxic soils".However, many of the measured phenotypes are likely to result from other stresses as well.It seems that additional work would be needed to identify Al-specific parameters, and some discussion on this may be needed.
These points include: 1. Provide appropriate details in the methods and figure legends as appropriate regarding both biological and technical replication (See Reviewer 1 comments).2. Expand the Discussion to cover the potentially unexpected observation that NPQ decreases with increasing AI concentration at the first two time points (MT1 and MT2), but these parameters both decrease at increasing Al concentration at MT3 and MT4.rETR shows a similar change in Al concentration response -in this case, increasing at MT1 and MT2, and decreasing with increasing Al concentration in MT3 and MT4.Although these opposite trends for both traits were reported in the Results they are not discussed or put in context of what is reported in the literature.Mechanistic insights in the Discussion would be useful.3. The authors conclude that these above-ground Al-induced phenotypes identified by highthroughput imaging phenotyping could be used "in remote sensing studies to map Al-toxic soils".However, many of the measured phenotypes are likely to result from other stresses as well.It seems that additional work would be needed to identify Al-specific parameters, and some discussion on this may be needed.
(3) NPQ Value Discrepancies: There is an inconsistency in the non-photochemical quenching (NPQ) values under different Al concentrations.Specifically, higher Al3+concentrations decrease NPQ in MT1 and MT2 but increase NPQ in MT3 and MT4.An explanation for these contrasting responses should be provided to better understand the underlying physiological mechanisms.
(4) There are errors in the units presented in the manuscript: Lines 136 and 141: The light unit should be written as "µmol m−2 s-1" instead of "µM m−2 s-1".
(5) In Figure 1, the treatment notation should be corrected to "400 µM Al" rather than "400 µM Al L-1".(6) The title of Table 1 should be placed above the table for consistency with standard formatting practices.

Reviewer #2:
The authors tried to design, perform, and present valuable research about "Quantifying Aluminium Toxicity Effects on Corn Phenotype Using Advanced Imaging Technologies".I have some comments and suggestions for the current manuscript.After carefully reading the manuscript, I recommend acceptance.Minor comments: L150 Please move the title of table 1 to the top of the table.Please check again the scientific names of plants and other cases in the reference section.I am pleased to inform you that your manuscript "Quantifying Aluminium Toxicity Effects on Corn Phenotype Using Advanced Imaging Technologies" has been accepted for publication in Plant Direct.Your article will appear online in the next available issue of Plant Direct.To ensure your article gets published as quickly as possible, please pay attention to the steps detailed below.We have found that most of the delays happen at this stage, especially at the payment stage, so please respond as quickly as possible when prompted.

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Gustavo MacIntosh
Editor, Plant Direct ---------------------------------------------------------------------------- "During the different measurements (from MT1 to MT4), chlorophyll fluorescence parameters NPQ, qN, фnpq, and rETR showed inconsistent behaviour.Namely, higher Al3+ concentrations decreased NPQ, qN, фnpq, and increased rETR in MT1 and the opposite, increased NPQ, qN, and фnpq, and decreased rETR in MT3 and MT4.Similarly, the exposure to cadmium toxicity caused a decrease in NPQ, the quantum of regulated energy dissipation, and the quantum yield of nonregulated energy dissipation in two Buddleja species (Gong et al., 2020).These results could be explained by increased antioxidative mechanisms, such as the Mehler-peroxidase pathway (Grace and Logan,1996) during the early stage of the stress.This decrease in non-photochemical quenching parameters and related electron consumption can be attributed to a reduction in the induction of NPQ, as well as a shift in the balance between the quickly and slowly reversible components of NPQ (Chen and Gallie, 2008).However, the specific sites and mechanisms of NPQ remain elusive (Nicol et al., 2019).In addition, the prolonged exposure to the Al toxicity caused an increase in NPQ and a decrease in rETR, indicating the exhaustion of these protective mechanisms.These findings suggest a complex interplay between antioxidative mechanisms and NPQ in plants, with implications for plant acclimation to Al stress." 3. The authors conclude that these above-ground Al-induced phenotypes identified by highthroughput imaging phenotyping could be used "in remote sensing studies to map Al-toxic soils".However, many of the measured phenotypes are likely to result from other stresses as well.It seems that additional work would be needed to identify Al-specific parameters, and some discussion on this may be needed.

Author response:
The paragraph which discusses the possible effect of other stresses causing similar phenotypic symptoms to those found under the Al toxicity has been added.Please see Line numbers 394-401.
"Our study indicates that Al toxicity in corn can be assessed very early (after four days of exposure) using non-destructive image-based technology.However, Al toxicity in plants can cause a variety of disorders, among others: root growth inhibition, nutrient uptake and transport disruption and oxidative stress (Bojórquez-Quintal et al., 2017;Kochian et al., 2015;Lidon and Barreiro, 2002;Zhang et al., 2019).Therefore, the phenotypic symptoms found under conditions of Al toxicity may be caused by complex interactions between the primarily toxic effect of aluminium and other physiological disorders that occur under conditions of acidic soil and Thank you very much for giving us an opportunity to review your work.I look forward to receiving the next version.
-Reviewer comments: Reviewer #1: Szőke et al. conducted a comprehensive analysis using image-analysis phenotyping to evaluate the responses of corn seedlings to different Al toxicity conditions.While the study provides valuable descriptive results based on a single genotype, it lacks mechanistic insights.Furthermore, several areas require clarification and correction: Comments:(1) The manuscript does not specify the number of replicates used in the different analyses.Please provide this information to ensure the robustness and reproducibility of the results.
MS TITLE: Quantifying Aluminium Toxicity Effects on Corn Phenotype Using Advanced Imaging Technologies Dear Dr. Boris Lazarevic: Editor comments The discussion about the NPQ and rETR has been added.Please see the Line number 366-382.Due to this new paragraph in the discussion, several new references are used and included in the list of references.Gong et al., 2020 (Line number 505); Chen and Gallie, 2008 (Line number 476); Grace and Logan,1996 (Line number 510), and Nicol et al., 2019 (Line number 577).
aluminium toxicity."Due to this new paragraph in the discussion, two new references were used and included in the list of references.Please see Bojórquez-Quintal et al., 2017 (Line number 465) and Zhang et al., 2019 (Line number 636).Also, the conclusions are slightly changed.Please see the Line numbers 408-414 Although this study was performed under controlled conditions, and the results should be confirmed under field conditions with combinations of different stresses, such as nutrient deficiencies and oxidative stress, it demonstrated the possibility of using selected phenotypic traits in remote sensing studies to map Al-toxic soils and/or in high-throughput phenotyping studies to screen Al-tolerant genotypes.4. Line 194: HDS should be HSD (Honestly Significant Difference) Author response:

: State in the legend or in table what values are being presented, presumably p-values.
: State in the legend or in table what values are being presented, presumably p-values.